In situ surface-enhanced Raman scattering (SERS) detection of trace analytes with high sensitivity, especially in complex aqueous solution environments such as polluted water and biological fluids, remains an urgent task. Herein, a micro-extraction SERS membrane was developed by folding a plasmonic structure composing Au@Ag nanoparticles (NPs) and CuO nanospikes (NSs) via capillary action. In the numerical simulation using COMSOL software, we demonstrated that this structure with a parallel facing state can effectively trap and utilize incident photons to excite multiple plasmon resonance couplings. Moreover, it detected the molecular aqueous solution via capillary action in only 5 min. Benefitting from the dense volumetric hotspots and the simple trace extraction solution method, we utilized the proposed membrane for ultrasensitive in situ SERS detections of microcystin–LR (5 ×10–6 μg/L) toxins in drinking water, adenosine biological fluid (10–10 M), and carcinogenic malachite green (10–11 M) in seafood using a portable Raman instrument. The findings of this study provide promising insights for achieving in situ SERS detection of trace analytes in complex aqueous environments.